The skin of mammals is their main defense barrier against external aggressions, whether they are chemical, mechanical or infectious aggressions. External aggressive agents also include environmental factors such as UV rays, tobacco smoke, pollution and climate. The skin has a cutaneous microbial flora forming its immune protection system; any unbalance in the population of said flora entails a functional immune deficit which often involves the invasion of the skin by autochthonous skin bacteria or by bacteria that are not usually in the skin, a process thus commencing which can result in a clinically established infection. Likewise, skin flora has multiple important functions of homeostasis, defense against bacterial infections (by interference), lipid degradation and production of volatile components responsible for body odor.
For example, the microorganisms which live as saprophytes in the surface of human skin, in its cracks, flakes, stratum corneum and hair follicles, have an important protective role as an additional skin barrier to the surface corneum and lipid layers, which determine the permeability between the internal and external medium. This dermal flora is formed by resident and transient microorganisms, and are bacteria, fungi and parasites.
Resident microorganisms have the capacity to multiply and survive adhered to the surface and are dominant skin constituents; examples of them are Corynebacterium bovis, C. mutissium, C. xerosis, C. hoffmani, Propionibacterium avidum, P. granulosum, Acinetobacter, the yeast Malassezia furfur, Pityrosporum ovale and P. orbiculares, as well as some groups of the Candida family, such as C. glabrata. The saprophyte parasite which is located in hair follicles, Demodex folliculorum, can be pathogenic.
Transient skin flora is mainly represented by Gram-positive bacteria, such as group A Streptococcus, Staphylococcus aureus and of the Neisseria genus or fungal flora such as Candida albicans, which is considered pathogenic whenever it is isolated in the skin.
Normal skin flora can be modified by several environmental factors, such as moisture and temperature, age, sex and race, as skin characteristics vary among people, which favors the colonization and proliferation of certain groups of microorganisms. Skin colonization depends on particular characteristics of each topographic area of the body, and the predominance of certain groups of microorganisms also varies according to the latter. In the scalp, for example, there is mixed flora, with bacteria, fungi and parasites, such as Pityrosporum ovale, Staphylococcus, Corynebacterium and Demodex folliculorum. Different groups of microorganisms of the axillary and perianal region, vulva or interdigital spaces can thus be isolated.
When this microbiological barrier is weakened or destroyed, as in the case of eczemas, irritations or aggressive skin treatments, pathogenic microorganisms can colonize skin or even traverse it, thus escaping the non-specific defense mechanisms of the skin. Thus, the presence of cutaneous microbial flora provides the skin with a defense barrier against pathogenic microorganisms by a nutritional competition phenomenon and by the secretion of substances with enzymatic and/or bactericidal activity.
The proliferation of pathogenic microorganisms such as for example Staphylococcus aureus, Streptopyogenes or Propionibacterium acnes or some yeasts entails a deregulation of the cutaneous flora system and can lead to more severe disorders or pathologies in the skin, mucosae, scalp and/or nails such as eczemas, candidiasis, dermatitis, onychomycosis or dermatosis among others. Likewise, in wound healing processes there is always a risk of infection, because the defense mechanisms of the skin, mucosae, scalp and/or nails are reduced. Wounds can be a result of physical injuries such as for example cuts, abrasions, burns, irritations, scrapes or exposure to chemical agents among others, a result of surgical processes such as for example surgical incisions or skin grafts among others, as well as a result of pathologies and even chronic conditions such as for example diabetic ulcers or venous ulcers among others. In a wound, the amount of inoculum of pathogenic microorganism, the virulence of said pathogens and the defense mechanisms of the host will determine if the wound will develop an infection, such that during healing processes the treatment with bactericidal compounds or compounds stimulating the defenses of the host are therapeutically useful [Edlich, R. F., Kenney J. G., Morgan R. F., Nichter L. S., Friedman H. I. and Rodeheaver G. T. (1986) “Antimicrobial treatment of minor soft tissue lacerations: a critical review” Emerg. Med. Clin. of North Am. 4:561-80].
In the same way, the growth of pathogenic microorganisms, and specifically the proliferation of Pseudomonas aeruginosa can also affect ocular mucosae, leading to ocular infections which can produce corneal ulcers. The risk of ocular infections is enhanced not only by bad hygiene habits, especially of the hands, but also by the daily use of contact lenses [Buehler P. O., Schein O. D., Stamler J. F., Verdier D. D. and Katz J. (1992) “The increased risk of ulcerative keratitis among disposable soft contact lens users” Arch. Ophthalmol. 110:1555-1558; Wilhelmus K. R. (1987) “Review of clinical experience with microbial keratitis associated with contact lenses” CLAO J. 13:211-214].
Some people have a specific risk of contracting infections in the skin, mucosae, scalp and/or nails because they have a suppressed immune system, such as for example AIDS patients or those people who are undergoing a chemotherapy or radiotherapy treatment due to cancerous processes [Epstein J. B. and Chow A. W. (1999) “Oral complications associated with immunosuppression and cancer therapies” Infect. Dis. Clin. North Am. 13:901-23]. In the same way, those people in stressful situations often have a suppressed immune system making them susceptible to contracting infections in the skin, mucosae, scalp and/or nails [Biondi M. and Zannino L. G. (1997) “Psychological stress, neuroimmunomodulation, and susceptibility to infectious diseases in animals and man: a review” Psychother Psychosom. 66:3-26].
Bromhidrosis or fetid odor is a result of microorganism proliferation in the skin, especially in the areas of the skin with a high degree of perspiration such as the axillae, genitalia or feet [Leyden J. J., McGinley K. J., Holzle E., Labows J. N. and Kligman A. M. (1981) “The microbiology of the human axilla and its relationship to axillary odor” J. Invest. Dermatol. 77:413-6]. The characteristic and unpleasant odor of sweat is a result of the decomposition of sweat and moist skin by bacteria and yeasts, and the consequent release of malodorous substances such as for example steroids, and can be treated with compounds controlling or reducing the microbial population of the area in which perspiration occurs [Elsner P. (2006) “Antimicrobials and the Skin Physiological and Pathological Flora” Curr. Probl. Dermatol. 33:35-41].
Another result of microorganism proliferation in the oral cavity is halitosis or malodor of the mouth. 85%-90% of the origin of halitosis is in oral causes such as the periodontal conditions, dentures and badly adapted restorations or a deficient dental hygiene. The microflora of the dorsal surface of the tongue and mostly Gram-negative anaerobic bacteria decompose food remains between teeth, remains of cells of the oral mucosa or of blood or of saliva, producing volatile substances such as simple fatty acids such as butyric acid, propionic acid or valeric acid and sulfurous components such as methyl mercaptan or hydrogen sulfide, or protein derivatives such as putrescine and cadaverine. The microorganisms causing halitosis include Treponema denticola, Prevotella intermedia, Porphyromonas gingivalis, Bacteroides forsythus, Fusobacterium periodonticum or Stomatococcus mucilaginus among others [De Boever E. H. and Loesche W. J. (1995) “Assessing the contribution of anaerobic microflora of the tongue to oral malodor” J. Am. Dent. Assoc. 126:1384-1393; De Boever E. H., De Uzeda M. and Loesche W. J. (1994) “Relationship between volatile sulfur compounds, BANA-hydrolyzing bacteria and gingival health in patients with and without complaints of oral malodor” J. Clin. Dent. 4:114-119; Kozlovsky A., Gordon D., Gelernter I., Loesche W. J. and Rosenberg M. (1994) “Correlation between the BANA test and oral malodor parameters” J. Dent. Res. 73:1036-1042]. To control the malodor of buccal origin the treatment must be aimed towards the elimination of these microorganisms, whereby compounds controlling or reducing the microbial population of the buccal area will be useful in the treatment of halitosis.
Likewise, pathogenic microorganism proliferation can be reinforced by the reduction of the natural defense systems of mammals, and specifically by the reduction of defensin expression, defensins being specific proteins against infections which are located in the skin and in mucosae.
Defensins are a class of natural antimicrobial peptides present in plants, insects and in different mammals, including human beings. They are small molecules of about 30-40 amino acids, having in common a large number of positively charged amino acids such as arginine, as well as the presence of cysteine residues forming disulfide bonds conferring them their three-dimensional structure containing a set of anti-parallel β sheets as a motif [Martin E., Ganz T. and Lehrer R. I. (1995) “Defensins and other endogenous peptide antibiotics of vertebrates” J. Leukocyte Biol. 58:128-133; Ganz T. and Lehrer R. I. (1994) “Defensins” Curr. Opinion Immunol. 6:584-589].
In mammals, defensins are classified into two families, according to the pattern of disulfide bonds that they have [Harder J., Bartels J., Christophers E. and Schröder J. M. (2001) “Isolation and characterization of human β-defensin-3, a novel inducible peptide antibiotic” J. Biol. Chem. 276:5707-5713]. In the case of human beings, α-defensins or HNPs have three disulfide bonds between the cysteine residues Cys1-Cys6, Cys2-Cys4 and Cys3-Cys5, and are located in neutrophils (HNP1 to HNP4) and in the gastrointestinal apparatus (HNP5 and HNP6). Human β-defensins or hBDs have three disulfide bonds between the cysteine residues Cys1-Cys5, Cys2-Cys4 and Cys3-Cys6, they are constitutively expressed in keratinocytes and are located in the kidney, pancreas, saliva, lungs, placenta and skin (hBD1), in the skin, trachea and lungs (hBD2), in the skin, trachea, tonsils and tongue (hBD3) and in the testicles and stomach (hBD4).
hBD2 and hBD3 are the only human defensins which are inducible and are regulated at transcriptional level in response to the contact with microorganisms. These hBDs are overexpressed by differentiated keratinocytes in those places in which an inflammation and/or an infection occurs [Harder J., Bartels J., Christophers E. and Schröder J. M. (1997) “A peptide antibiotic from human skin” Nature 387:861]. The mechanism of action proposed for hBD2 is the binding to the target bacteria and its subsequent insertion in the lipid membrane of the microbe, altering the permeability of the membrane and therefore its internal homeostasis. hBD2 is highly effective killing Gram-negative bacteria, whereas it only has bacteriostatic activity against Gram-positive bacteria. The spectrum of hBD3 is broader than that of hBD2 and is effective as a bactericide against different Gram-positive and Gram-negative bacteria [Harder J., Bartels J., Christophers E. and Schröder J. M. (2001) “Isolation and characterization of human β-defensin-3, a novel inducible peptide antibiotic” J. Biol. Chem. 276:5707-5713; Garcia J. R., Jaumann F., Schukz S., Krause A., Rodriguez-Jimenez J., Forssmann U., Adermann K., Kluver E., Vogelmeier C., Becker D., Hedrich R., Forssmann W. G. and Bals R. (2001) “Identification of a novel, multifunctional β-defensin (human β-defensin 3) with specific antimicrobial activity. Its interaction with plasma membranes of Xenopus oocytes and the induction of macrophage chemoattraction” Cell Tissue Res. 306:257-264].
There is a direct correlation between hBD expression and the incidence of infections in human beings. hBD1 and hBD2 are extensively expressed in oral inflammation tissue samples, as well as in primary oral keratinocytes [Harder J., Bartels J., Christophers E., and Schröder J. M. (2001) “Isolation and characterization of human β-defensin-3, a novel inducible peptide antibiotic” J. Biol. Chem. 276:5707-5713]. In addition, the skin of patients affected by psoriasis, in which epithelial hBDs are overexpressed, has relatively low infection statistics, whereas in patients with atopic dermatitis, in whom hBD expression is suppressed, injuries are easily infected [Nomura I., Goleva E., Howell M. D., Hamid Q. A., Ong P. Y., Hall C. F., Darse M. A., Gao B., Boguniewicz M., Travers J. B. and Leung D. Y. (2003) “Cytokine milieu of atopic dermatitis, as compared to psoriasis, skin prevents induction of innate immune response genes” J. Immunol. 171:3262-3269; Ong P. Y., Ohtake, T., Brandt C., Strickland I., Boguniewicz M., Ganz T., Gallo R. L. and Leung D. Y. (2002) “Endogenous antimicrobial peptides and skin infections in atopic dermatitis” N. Engl. J. Med. 347:1151-1160].
The pharmaceutical industry has focused its efforts on the development of a potent pharmacological collection of compounds with bactericidal and/or fungicidal activity to treat infections of the skin, mucosae, scalp and/or nails. Said treatments are not free of side-effects and further have the drawback that their continuous use leads to the resistance of pathogenic microorganisms against said compounds. It is therefore necessary to develop compounds which allow controlling pathogenic microorganism proliferation in the skin, mucosae, scalp and/or nails in a more naturally, safely and effectively way.
A valid alternative to the classic treatment with bactericidal and/or antifungal compounds is the induction of the endogenous defense systems of organisms and, specifically, the induction of endogenous β-defensin expression. The state of the art describes that hBD2 and hBD3 expression is inducible, and can be stimulated by means of bacteria or yeast extracts [Harder J., Bartels J., Christophers E. and Schröder J. M. (1997) “A peptide antibiotic from human skin” Nature 387:861], by isoleucine [Fehlbaum P., Rao M., Zasloff M. and Anderson G. M. (2000) “An essential amino acid induces epithelial β-defensin expression” PNAS 97:12723-12728] or by alkylamines [Bukowski J. F., Morita C. T. and Brenner M. B. (1999) “Human gamma delta T cells recognize alkylamines derived from microbes, edible plants, and tea: implications for innate immunity” Immunity 11:57-65].
Different patents and applications describe the use of plant extracts as agents inducing β-defensin expression. Application FR 2,843,125 A1 of Coletica S. A. and YSL Beauté describes the use of certain plant extracts, among them boldo extract, as well as the use of vitamin A and its precursors, α-MSH and its peptide fragments or analogs, calcium and its salts or isoleucine esters as hBD production stimulators, with the compromise that they do not stimulate pro-inflammatory molecule production, and their application in the cosmetic and pharmaceutical field as antifungal or antibacterial agents. International application WO 2005/077349 A1 of Otsuka Pharmaceuticals Co. describes the use of different plant extracts, protein hydrolysates, amino acids, enzymes or proteins as agents inducing hBD synthesis and their use in the cosmetic, food or pharmaceutical field. Patent application JP 2005-270117 A of Morinaga Co. also describes the use of different plant extracts as agents stimulating hBD expression.
Other documents such as patent application US 2004/0259795 A1 of Gemma Biotechnology Ltd. describe the use of proteins such as the CD14 milk protein or fragments thereof as agents stimulating defensin synthesis and their application as a medicinal product or dietary agent for reducing sepsis symptoms.
The state of the art also describes certain types of small molecules which are capable of inducing hBD synthesis. International application WO 01/68085 A1 of Genaera Corp. and patent application US 2002/0076393 A1 of Magainin Pharmaceuticals Inc. describe the use of the amino acids isoleucine or valine, their stereoisomers and some analogs of said amino acids in the treatment or prevention of infectious processes through the induction of hBD expression. Likewise, application EP 1,671,629 A1, also of Otsuka Pharmaceuticals Co., describes the use of certain organic acids, specifically fumaric, malic, citric, ascorbic, lactic, acetic, adipic, tartaric, cinnamic, glutamic or succinic acids as hBD expression inducers. International patent application WO 2005/115403 A2 of the Cedars Sinai Medical Center describes a method for treating a condition comprising the administration of vitamin D3 or its analogs as endogenous hBD production stimulators, and its use in the pharmaceutical or dermopharmaceutical field. Patent application FR 2,896,691 A1 of Pierre Fabre Dermo-Cosmetique S. A. describes the use of alkylglucoside esters as hBD expression inducers and their use in the dermatological or dermocosmetological field.
In the same way, it is known in the state of the art that some microorganisms or microorganism extracts have an efficacy inducing endogenous hBD expression. Patent application US 2005/0196480 A1 of Estee Lauder Companies describes the use of Lactobacillus extracts as hBD production stimulators, and their application in the cosmetic field for the reduction of skin microflora, the treatment of acne and the reduction of sensitivity of sensitive skin. International patent application WO 2004/055041 A2 of Case Western Reserve University describes a defensin-stimulating composition comprising a 12 kDa defensin-inducing peptide associated to fusobacterium and its use for the treatment of infections caused by the human immunodeficiency virus. Application FR 2,879,452 A1 of L′Oreal describes the hBD synthesis-inducing capacity of a non-photosynthetic, non-fructifying filamentous bacterium, and its use in the cosmetic field. Patent U.S. Pat. No. 6,984,622 B2 of The Regents of the University of California describes the use of lipopolysaccharide or its fragments as agents stimulating hBD expression for the treatment or prevention of ocular infections and of ocular wounds. International application WO 2007/020884 A1 of Meiji Dairies Corp. describes the use of bifidobacteria and lactic acid bacteria extracts for the prevention of infections by increasing endogenous 13-defensin levels. Patent application JP 2006-241023 A of Asahi Breweries Ltd. describes the use of mannose-rich yeast as an agent inducing defensin synthesis and its use in the pharmaceutical and food field.
None of the documents known in the state of the art describes peptides not derived from natural products capable of inducing hBD expression.